Monoclonal antibody recognizing phosphatidylinositol-3,4,5-triphosphate

ABSTRACT

A novel monoclonal antibody that specifically recognizes phosphatidylinositol-3,4,5-triphosphate (PIP3) but does not cross-react with structurally similar phospholipid antigens is advantageous for PIP3-specific immunoassay. The gene in the variable regions of the monoclonal antibody has been identified, which enables producing recombinant antibodies.

FIELD OF THE INVENTION

[0001] The present invention relates to a monoclonal antibody tophosphatidylinositol-3,4,5-triphosphate. The present invention alsorelates to a method for immunoassay ofphosphatidylinositol-3,4,5-triphosphate using the monoclonal antibody.

BACKGROUND OF THE INVENTION

[0002] The basic system for survival of multicellular organisms,including humans, includes a “cellular signal transduction system,” inwhich an extracellular signal is transferred to the inside of the cellto induce a necessary cell response. It is becoming clear thatabnormality in this system causes pathological conditions attributed toan inadequate cell response, such as neoplastic transformation of cells.Phosphatidylinositol (hereinafter abbreviated as PI), which is aphospholipid present in various biomembranes, changes the phosphorylatedstate of the inositol ring by the action of enzymes activated byextracellular stimulation to transduce an extracellular signal intocells. Phosphatidylinositol-3,4,5-triphosphate with a phosphorylatedinositol group at the 3-position has been shown, inter alia, to beproduced from phosphatidylinositol-4,5-biphosphate (hereinafterabbreviated as PI-4,5-P2) by the action ofphosphatidylinositol-4,5-kinase (hereinafter abbreviated as PI3K)activated by stimulating various proliferation or differentiationfactors and to participate not only in signal transduction into the cellnucleus but also in versatile and fundamental cell responses such asconstruction of cell backbone and vesicular transport of substances(Fukui, Y. et al., J. Biochem., 124, 1-7, 1998).Phosphatidylinositol-3,4,5-triphosphate used herein is abbreviated PIP3;if it is necessary to specify the phosphorylated position, thetriphosphate is designated PI-3,4,5-P3. The structure of PIP3 isschematically shown below.

[0003] Studies on the kinetics of PIP3 both in cells and in livingorganisms help clarify the fundamental cellular signal transduction andshed light on pathological conditions of various diseases. The role ofPIP3 in cells has been explored so far mainly by indirect means of usingPI3K inhibitors or modifiers such as activated or inactivated PI3K. As adirect approach, a quantitative assay has been established for PIP3.However, the known assay for PIP3 requires using a radioisotope, whichnecessitates special facilities for the assay. Moreover, the assayitself is time-consuming due to complicated operations includingextraction and chromatography in many steps. Therefore, a simple assaytechnique has been desired.

[0004] For instance, antibodies that can recognize PIP3 will beimportant tools for further investigations. These antibodies will beuseful for purification and immunoassay of PIP3 or as inhibitors againstPIP3. However, phospholipid antigens are generally known to have a poorantigenicity. It is thus difficult to obtain antibodies to the antigens.PIP3 has an additional problem in that it is difficult to produce inlarge quantities. Though attempts to produce the antibodies usingelaborated adjuvants were reportedly successful, most of the antibodiesproduced have a poor ability to recognize the phosphorylated state ofPI. In most cases, it is difficult to distinguish PIP3 from PI orPI-4,5-P2 due to their cross-reactivity.

[0005] It has been reported that a rabbit was immunized to produce apolyclonal antibody to PI-4,5-P2 (Molec. Immun., 16, 193-196, 1979). Thepolyclonal antibody exhibited cross-reactivity with other phospholipidantigens such as phosphatidylinositol-4-phosphate (hereinafterabbreviated as PI-4-P) or cardiolipin (hereinafter abbreviated as CL). Amonoclonal antibody to PI-4-P was obtained by immunization withliposomes (Molec. Immun., 21, 863-868, 1984). However, it was confirmedthat the antibody was also cross-reactive with other phospholipids, thatis, the cross-reactivity with PI-4,5-P2 was observed by thecomplement-dependent liposome lysis assay.

[0006] A monoclonal antibody to PI-4,5-P2 was produced using as animmunogen a suspension of PI-4,5-P2 together with phosphatidylcholine ormethylated BSA (Proc. Japan Acad., 59, 28-32, 1983). The thus-producedmonoclonal antibody has been verified to be cross-reactive with anacidic phospholipid such as PI-4-P or PI. Furthermore, a monoclonalantibody specific to PI-4,5-P2 and showing a low cross-reactivity withother phospholipids was obtained using a Salmonella cell as an adjuvant(Miyazawa A., Molec. Immun., 26, 1025-1031, 1988). However, there hasbeen no report of an antibody highly specific to PIP3 andnon-cross-reactive with other phospholipid antigens being obtained. Forthis reason, no immunoassay technique for PIP3 has been found yet.Immunoassay is an excellent technique that can achieve a highsensitivity and accuracy in a simple operation. Accordingly, animmunological assay for PIP3 is strongly desired for further studies oncellular signal transduction.

SUMMARY OF THE INVENTION

[0007] An objective of the present invention is to provide an antibodyspecifically recognizing PIP3. Another objective of this invention is toprovide an immunological assay method using the antibody. Morespecifically, the present invention seeks to provide a novel antibodyspecifically recognizing PIP3 and a simple method for determining PIP3with high sensitivity, like enzyme immunoassay, without requiring anyspecial facilities.

[0008] Producing an anti-PIP3 antibody is a problem because it isdifficult to obtain a large quantity of antigens and the poorantigenicity of phospholipids used as antigens makes it difficult toproduce an antibody of high titer. The inventors have solved the formerproblem by chemical synthesis of PIP3 that enables it to be produced inlarge quantities. The inventors have also overcome the problem of poorantigenicity by enhancing the antigenicity, using an immunogen obtainedthrough adsorption of PIP3 to dead Salmonella cells. In this way, theinventors have succeeded in producing a novel monoclonal antibody thatbinds specifically to PIP3. Using the antibody, an immunological assayspecific to PIP3 in the living organism can be performed successfully.Furthermore, the inventors have isolated a gene encoding the amino acidsequence that constitutes variable regions of the antibody and havedetermined the nucleotide sequence, which will enable producingrecombinant antibodies. The inventors have also found that topologicalPIP3 distribution in cells can be identified and inhibitors specific tothe function of PIP3 can be developed, using the antibody of the presentinvention.

[0009] Specifically, the present invention relates to the followingantibody and variable regions thereof as well as an immunological assaymethod using the antibody.

[0010] (1) An antibody specifically recognizingphosphatidylinositol-3,4,5-triphosphate.

[0011] (2) An antibody of (1), wherein the antibody is a monoclonalantibody.

[0012] (3) An antibody of (2), which recognizes an antigenic determinantcomprising an inositol group and a glycerol backbone inphosphatidylinositol-3,4,5-triphosphate.

[0013] (4) An antibody of any one of (1) through (3), which isnon-cross-reactive with phosphatidylinositol-4,5-diphosphate.

[0014] (5) A variable region of immunoglobulin heavy chain specificallybinding to phosphatidylinositol-3,4,5-triphosphate, comprising an aminoacid sequence shown by SEQ ID NO:2 or an amino acid sequence of SEQ IDNO:2 in which one or more amino acid residues have been substituted,deleted or added.

[0015] (6) A variable region of immunoglobulin light chain specificallybinding to phosphatidylinositol-3,4,5-triphosphate, comprising an aminoacid sequence shown by SEQ ID NO:4 or an amino acid sequence of SEQ IDNO:4 in which one or more amino acid residues have been substituted,deleted or added.

[0016] (7) CDR1 in immunoglobulin heavy chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:5 or an amino acid sequence of SEQ ID NO:5in which one or more amino acid residues have been substituted, deletedor added.

[0017] (8) CDR2 in immunoglobulin heavy chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:6 or an amino acid sequence of SEQ ID NO:6in which one or more amino acid residues have been substituted, deletedor added.

[0018] (9) CDR3 in immunoglobulin heavy chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:7 or an amino acid sequence of SEQ ID NO:7in which one or more amino acid residues have been substituted, deletedor added.

[0019] (10) CDR1 in immunoglobulin light chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:8 or an amino acid sequence of SEQ ID NO:8in which one or more amino acid residues have been substituted, deletedor added.

[0020] (11) CDR2 in immunoglobulin light chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:9 or an amino acid sequence of SEQ ID NO:9in which one or more amino acid residues have been substituted, deletedor added.

[0021] (12) CDR3 in immunoglobulin light chains specifically binding tophosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO:10 or an amino acid sequence of SEQ ID NO:10in which one or more amino acid residues have been substituted, deletedor added.

[0022] (13) An immunogen composition for use in producing an antibody ofany one of (1) through (4), comprising a mixture of a dead Salmonellacell as an adjuvant and phosphatidylinositol-3,4,5-triphosphate.

[0023] (14) A method for producing an antibody of any one of (1) through

[0024] (4), which comprises immunizing an immunogen composition of (13).

[0025] (15) An immunoassay method which comprises the steps of reactingthe antibody of any one of (1) through (4) or a variable region thereofwith phosphatidylinositol-3,4,5-triphosphate present in a sample, anddetecting the binding based on an immunological reaction between theantibody or a variable region thereof and the triphosphate.

[0026] (16) An immunoassay method of (15), which comprises observing thedegree to which the immunological reaction between the antibody or avariable region thereof and an antigenic determinant recognized therebyis inhibited by phosphatidylinositol-3,4,5-triphosphate present in asample.

[0027] (17) A kit for immunoassay forphosphatidylinositol-3,4,5-triphosphate comprising the antibody of anyone of (1) through (4) or a variable region thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 shows graphs showing the results of indirect enzyme-linkedimmunosorbent assay (hereinafter referred to as ELISA) performed toverify the cross-reactivity of AP3-11 with various inositolpolyphosphates. The ordinate designates the binding (%) of the antibodyAP3-11, and the abscissa designates the concentration (Log ng/mL) ofcompetitive compounds.

[0029]FIG. 2 shows graphs showing the results of liposome lysis assayperformed to verify the binding activity of the antibody AP3-11 withvarious phospholipids. The ordinate designates the lysis (%) ofliposomes, and the abscissa designates the concentration (pg/mL) of theantibody.

[0030]FIG. 3 shows graphs showing the results of liposome lysis assayperformed to verify the binding activity of the antibody AP3-11 withvarious phosphatidylinositol polyphosphates. The ordinate designates thelysis (%) of liposomes, and the abscissa designates the concentration(pg/mL) of the antibody.

[0031]FIG. 4 shows graphs showing the results of ELISA performed toverify the binding activity of the antibody AP3-11 with PIP3 andPI-3,4-P2. The ordinate designates absorbance, and the abscissadesignates the concentration (pg/mL) of the antibody.

[0032]FIG. 5 shows graphs showing the results of indirect ELISAperformed to verify the cross-reactivity of AP3-11 with various inositolpolyphosphates. The ordinate designates absorbance, and the abscissadesignates the concentration (nM) of polyphosphate compounds which arecompetitive compounds.

[0033]FIG. 6 shows photographs showing the results of CBB staining onSDS-PAGE indicating the binding activity of the antibody AP3-11 withvarious PIP3 analogs, in which the numerical figures designate theamount (μM) of PIP3 added for absorption of the antibody.

[0034]FIG. 7 schematically shows the epitope site of the antibodyAP3-11.

[0035]FIG. 8 schematically shows a predicted hyper variable region(complementarity determining region (CDR)) in the variable region ineach of the light chains and the heavy chains of the antibody AP3-11.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The antibody of the present invention specifically recognizesPIP3 but is not cross-reactive with PI-4,5-P2. Throughout thespecification, the term “specific to PIP3” means that the antibody canrecognize the phosphorylated state of PIP3 and immunologicallydistinguish PIP3 from other phosphorylated compounds. The antibody ofthe present invention can be produced by the following immunologicalprocedures. PIP3 is suspended together with the killed Salmonella cells,and the resulting suspension is used as an immunogen. In more detail,lipopolysaccharides are removed from the dead Salmonella cells (GalanosC., Eur. J. Biochem., 24, 116-122, 1971). PIP3 is then coated onto thedead cells to prepare a suspension for use as an immunogen (Umeda M., J.Immun., 137, 3264-3269, 1986). PIP3 may be purified from cells orchemically synthesized. Chemical synthesis of PIP3 is known anddescribed in Sawada T., Bioorganic & Medicinal Chemistry Letters, 5,2263-2266, 1995. An animal suitable for immunization is immunized withPIP3. When an increase in the antibody titer is confirmed, the antibodyis recovered. The antibody may be polyclonal or monoclonal. Themonoclonal antibody is more advantageous in that an antibody with ahigher specificity can be selected. Furthermore, when the monoclonalantibody is established, it enables cloning cDNA coding for the aminoacid sequences that constructs the variable regions of the monoclonalantibody having a desired binding activity, as will be described later.

[0037] The monoclonal antibody can be produced by cloningantibody-producing cells. In general, antibody-producing cells recoveredfrom the immunized animal are subjected to cell fusion with anappropriate fusion partner. The resulting hybridomas are then screenedin terms of the activity of the produced antibodies (Gulfre G., Nature,266, 550-552, 1977). When mice are employed as the animal forimmunization, mouse-derived myeloma cells such as P3-X63-Ag.653 areadequate as the fusion partner. The hybridomas subjected to HATselection are screened first in terms of the binding activity to PIP3.The hybridomas producing the antibodies that have the binding activityto PIP3 are then subjected to a cross-reactivity test. In this test, thebinding activity to other phospholipid antigens is examined to screenhybridomas having an acceptable cross-reactivity. Acceptablecross-reactivity means a cross-reactivity that can be disregarded forthe desired use of the antibody. When the monoclonal antibody isemployed for an immunological assay, it has no substantialcross-reactivity if a signal provided by the cross-reactivity is reducedto the level of the background in the final assay system.

[0038] ELISA or liposome lysis assay is useful for verifying thereactivity of PIP3 or the cross-reactivity with other phospholipidantigens. In ELISA, microtiter plates sensitized with an antigen whosereactivity is to be observed are prepared. A sample solution obtained bysuitably diluting the hybridoma supernatants is then added to the wellsof the microtiter plates to initiate a reaction. After a thoroughreaction, the wells are washed, and a second antibody to immunoglobulinis then added for further reaction. The second antibody finally bound toeach well is assayed. Thus, the binding activity of the antibody presentin the culture supernatant to the antigen can be quantitativelydetermined. ELISA has been demonstrated for an antibody using aphospholipid antigen (Umeda M., J. Immun., 136, 2562-2567, 1986).

[0039] The liposome lysis assay utilizes the phenomenon that when anantibody reacts with an antigen-sensitized liposome, the liposome lysesby the action of the complement. Since the action of the complement isutilized, the technique is called complement-dependent liposome lysisassay. Liposomes comprise phospholipid antigens to be examined inaddition to dicetyl phosphate (hereinafter abbreviated as DCP),dimyristoyl phosphatidylcholine (hereinafter abbreviated as DMPC), andcholesterol. These lipid components are dissolved in an appropriateorganic solvent. The solution is then dried to prepare a lipid film.When the film is added to an aqueous solvent and the mixture is agitatedvigorously, liposomes of multilamellar structure are formed. In theliposomes thus prepared, phospholipid antigens are taken up asmembrane-constructing components. For this reason, an antigenicstructure close to the antigen present in actual cell membranes ispresented. It is thus appropriate for screening the monoclonal antibody.For easy screening, a fluorescent dye can be enclosed in the liposomesas a lysis marker. Typical examples of the fluorescent dye include4-methylumbelliferyl phosphate and calcein. When the antibody binds tothis liposome-constructing phospholipid antigen in the presence of itscomplement, the liposome is broken down to release the fluorescent dyein the liposome. This phenomenon is observed as increased fluorescenceintensity in the liquid phase. Complement-dependent liposome lysis assayusing phospholipid antigens such as PI-4,5-P2 is known (Molec. Immun.,26, 1025-1031, 1988). The phospholipids used to verify thecross-reactivity are phospholipid antigens having a similar structure.The cross-reactivity should be verified with analogous substances havinga similar partial structure. Specific examples of such compounds aregiven below, and their structural characteristics will be shown inexamples below. PC phosphatidylcholine PS phosphatidylserine PAphosphatidic acid PI phosphatidylinositol CL cardiolipin PEphosphatidylethanolamine PI-4,5-P2 phosphatidylinositol-4,5-biphosphateIP3 1,4,5-inositol triphosphate IP4 1,3,4,5-inositol tetraphosphate IP61,2,3,4,5,6-inositol hexaphosphate

[0040] In both ELISA and liposome lysis assay, the cross-reactivity ofthe antibody with other phospholipid antigens can be verified by thereaction system using PIP3 as an antigen. That is, to the reactionsystem containing PIP3 and the antibody to be examined for itsspecificity are added other antigens to be examined for thecross-reactivity with the antibody and the competitive reaction is thenobserved to confirm the cross-reactivity. This technique for verifyingthe cross-reactivity by means of the competitive inhibition is usefulfor rapid screening since it is unnecessary to prepare the reactionsystem for all antigens.

[0041] The procedures described above can yield an antibody of thepresent invention that has binding activity to PIP3 and canimmunologically distinguish PIP3 from structurally similar antigens suchas PI-4,5-P2.

[0042] The present invention further provides the amino acid sequencesconstituting the variable regions of the novel antibody having a desiredbinding activity to PIP3 and the nucleotide sequences encoding the same.More specifically, the present invention provides the immunoglobulinvariable regions containing the amino acid sequences shown by SEQ IDNO:2 and SEQ ID NO:4. The present invention further provides cDNAencoding the immunoglobulin variable regions containing the nucleotidesequences shown by SEQ ID NO:1 and SEQ ID NO:3. SEQ ID NO:1 and SEQ IDNO:2 are deduced from the heavy chain and, SEQ ID NO:3 and SEQ ID NO:4from the light chain, in the immunoglobulin molecule. These amino acidsequences or cDNA nucleotide sequences are not necessarily identical butmay vary so long as the specific binding activity to PIP3 is maintained.As will be later described, particularly the site corresponding to CDRis highly variable. In the CDR region, even amino acids may vary someoccasions.

[0043] In general, each immunoglobulin molecule consists of heavy chainshaving a larger molecular weight and light chains having a smallermolecular weight. The heavy and light chains each carries a regioncalled “a variable region” in about 110 amino acid residues at theN-terminus, which are different between the molecules. Variable regionsof a heavy chain and a light chain are designated VH and VL,respectively. The antigen-binding site is formed by forming a dimerthrough electrostatic interaction between the heavy chain variableregion VH and the light chain variable region VL. The variable regionconsists of three complementarity determining regions (CDRs) and fourframeworks. The CDR forms a complementary steric structure with theantigen molecule and determines the specificity of the antibody. Thethree CDRs inserted between the four framework regions (FRS) are presentlike a mosaic in the variable region (E. A. Kobat et al., Sequences ofproteins of immunological interest, vol. I, 5th edition, NIHPublication, 1991). The amino acid sequences of FRs are well conserved,but those of CDR are highly variable and may thus be calledhypervariable regions. Among the amino acid sequences of the antibodyspecifically recognizing PIP3, a CDR that determines the bindingactivity to antigens has been clarified in the present invention. Thus,the present invention further provides the CDR shown below (numberingthe N-terminal amino acid residue as 1 in SEQ ID NO:1 or 3), in whichthe number in parentheses corresponds Heavy Chain Light Chain CDR1 26-32(5) 24-34 (8) CDR2 52-56 (6) 50-56 (9) CDR3 99-109 (7)   89-97 (10)

[0044] The cDNAs bearing the nucleotide sequences coding the variableregions in immunoglobulin molecules can be cloned from hybridomas thatproduce the monoclonal antibody to PIP3. More specifically, PCR isperformed using the signal sequence of the gene in the variable regionsand the nucleotide sequence in the constant regions. The amplifiedproduct is introduced into an appropriate cloning vector for furtheramplification to produce a library of variable genes. Since the sitecorresponding to CDR will be a sequence specific to the variable regionsof the present invention, positive clones are screened from the libraryof the variable regions using the site as a probe. The resulting cDNA isinserted into a phage by linking the light and heavy chain variableregion genes through an appropriate linker. Thus, the cDNA may beexpressed as a single-stranded antibody (so-called ScFV). Alternatively,known immunoglobulins are inserted into a vector for expression toutilize the same for producing immunoglobulins. Examples of the vectorfor expressing immunoglobulins without limit include a SV40 virus-basedvector and a BPV (papilloma virus )-based vector. For example, BCMGS Neovector, one of the BPV vectors (Hajime Karasuyama, “Bovine PapillomaVirus Vector” from the extra issue of JIKKEN IGAKU (ExperimentalMedicine): IDENSHI KOGAKU (Genetic Engineering) Handbook, edited byMasami Muramatsu & Hiroto Okayama, Yodosha Publishing Co., pp. 297-299,1991), is desirable since the vector is transformed to COS7 cells toexpress a foreign gene efficiently.

[0045] Alternatively, the specificity of the antibody of the presentinvention may also be artificially reconstructed by incorporating theCDR into the framework of an optional immunoglobulin molecule. Such atechnique is called the CDR grafting antibody technique (P. T Jones etal., Nature, 321, 522, 1986) and has already been established forhumanizing mouse immunoglobulins. The CDRs of the present inventioninclude not only those completely identical but also variants so long asthe specificity to PIP3 is maintained. That is, the CDR amino acidsequences in which one or more amino acid residues are modified may alsobe used as the CDR sequence. The modified amino acid residues in theamino acid sequences of the CDR variant are preferably 30% or less, morepreferably 20% or less, most preferably 10% or less, within the entireCDR. Any FR can be used as the FR into which the CDRs are to beincorporated. The CDRs of the present invention are originally derivedfrom mouse immunoglobulins. However, the CDRs may be inserted into FRsof not only mouse immunoglobulins but also immunoglobulins of otherspecies. The cDNAs encoding the variable regions thus constructed may beexpressed by incorporating the same into the vectors described above.

[0046] In introducing mutations into CDRs, the above-described phagevector may be employed. The phage vector can express the antibodyactivity rapidly and hence can rapidly screen mutants. Moreover, thephage vector expresses the antibody molecule on the surface of hostbacteria in an amount sufficient for screening. A mutation introducedinto the CDRs remarkably increases the antibody binding activity. Thus,a single-stranded antibody having an improved binding activity can beproduced using the CDRs of the present invention.

[0047] The variable regions and CDR-incorporated variable regionsprovided by the present invention may be expressed in their originalforms. Alternatively, these variable regions may be expressed ascomplete immunoglobulin molecules by linking to a gene encoding theconstant regions.

[0048] To express variable regions by the cDNA-incorporated vector or bythe vector bearing the insert obtained by linking CDR alone to a certainFR, a dimer of the heavy and light chains can be produced by expressingheavy chain variable regions and light chain variable regions in thesame host cell. This can be done by co-transformation of a host cellwith a light chain expression vector and a humanized heavy chainexpression vector. The antibody of the present invention can be producedfrom the transformant. Preferred examples of the host for thetransformation include Chinese hamster ovary (CHO) cells (A. Wright & S.L. Morrison, J. Immunol., 160, 3393-3402, 1998) and SP2/0 cells (mousemyeloma) (K. Motmans et al., Eur. J. Cancer Prev., 5, 512-519,1996; R.P. Junghans et al., Cancer Res., 50, 1495-1502, 1990). Thetransformation can be performed by the lipofectin method (R. W. Maloneet al., Proc. Natl. Acad. Sci. USA, 86, 6077, 1989, P. L. Felgner etal., Proc. Natl. Acad. Sci. USA, 84, 7413, 1987), the electroporationmethod, the calcium phosphate method (F. L. Graham & A. J. van der Eb,Virology, 52, 456-467, 1973), or the DEAE-Dextran method.

[0049] When the expressed variable region is accompanied by the constantregion, the expression product may be purified through a protein Acolumn, a protein G column, an anti-immunoglobulin antibody affinitycolumn, etc. to recover the product as a purified protein. When only thevariable region is expressed, these techniques for purification do notapply. In that case, other suitable purification methods should beselected. For example, if the variable region is expressed as theproduct fused to a protein such as a histidine tag at the C-terminus,then the expression product is purified by affinity chromatography usingthe corresponding ligand.

[0050] According to the present invention, PIP3 can be immunologicallyassayed using the thus produced monoclonal antibody or variable regionsthereof. Immunological assay of PIP3 was impossible by conventionalmethods since an antibody itself specific to PIP3 was not available. Theantibody of the present invention provides excellent specificity to PIP3and hence provides an ideal immunological method for assaying PIP3.

[0051] According to the present invention, PIP3 can also be assayed byobserving the degree of PIP3 inhibition, utilizing the phenomenon thatthe binding between PIP3 and the antibody of the present invention(including the variable regions) is inhibited by PIP3 originating from asample to be analyzed. One inhibition assay that realizes such an assayprinciple utilizes immobilized PIP3. In more detail, PIP3 is adsorbedonto a container like a microtiter plate. A sample solution is added tothe plate. PIP3 can be physically adsorbed onto the container wall afterit is dissolved in an appropriate carrier such as phosphatidylcholine.The antibody of the present invention is then added, causing acompetitive reaction between PIP3 adsorbed on the container and PIP3 inthe sample solution with the antibody of the present invention. Theantibody bound (or unbound) to the solid phase can be readily assayed bylabeling the antibody with an appropriate marker. The quantity of PIP3present in the sample solution can then be determined by comparison withthe results obtained from a standard solution. The antibody can belabeled with a marker such as an enzyme, a fluorescence or aluminiferous substance. PIP3 can be assayed in a biological samplesolution such as a tissue, a cultured cell, or a body fluid like bloodor serum. The foregoing competitive reaction may also be performed byimmobilizing the antibody of the present invention onto the wall of acontainer. In this case, the labeled PIP3 is reacted with the antibodyconcurrently with a PIP3-containing sample.

[0052] The sample for the immunological assay may be either liquid orsolid. For example, a tissue specimen is immunologically stained toobserve the presence or absence of PIP3 or localization of PIP3. In apreferred embodiment, the antibody of the present invention recognizesthe epitope formed by the inositol group and the glycerol backbone ofPIP3. Since the epitope is assumed to be exposed on the surface of thecell membrane, the antibody is useful for staining a tissue specimen. Inthis case, localization of several phospholipids may be observed in thesame sample by using the antibody in combination with another antibodyspecifically recognizing a phospholipid, e.g., PI-4,5-P2. A knowndouble-staining technique involves staining the same sample usingdifferent antibodies each labeled with fluorescent dyes having differentwavelengths.

[0053] The present invention further provides a kit for use in theimmunoassay described above. More specifically, the kit of the presentinvention comprises the antibody of the present invention, a substraterequired for detecting the label, positive control, negative control,and a buffered solution used for diluting and washing a sample.

[0054] The present invention provides a monoclonal antibody thatspecifically binds to PIP3. The present invention further provides animmunological assay method using the antibody. The experimental resultsrevealed that the antibody recognizes, as the epitope, not only theinositol group but also the glycerol backbone of PIP3. Thus, theantibody of the present invention can distinguish PIP3 from otherinositol compounds.

[0055] The present invention further provides the gene encoding thevariable regions of the antibody and hence enables producing recombinantantibodies. Since the antibody of the present invention is highlyspecific to PIP3, the location of PIP3 in cells can be identified.Alternatively, signal transduction by PIP3 to the downstream can beinhibited using the specificity of the antibody to PIP3 to investigateany affect caused. The present invention facilitates conducting studiesthat could not be conducted by conventional assay methods.

[0056] The present invention will be described below in more detail withreference to examples. However, this invention is not to be construed tobe limited to those examples.

EXAMPLE 1 Producing Anti-PIP3 Monoclonal Antibody

[0057] To produce anti-PIP3 antibody, synthesized PIP3 was coated ontodead Salmonella cells as an adjuvant. The coated cells were then used asan immunogen. Namely, Salmonella minnesota was cultured overnight torecover the cells. The cells were centrifuged and washed twice withdistilled water and once with diethyl ether, then dried in vacuo. Thecells were then dispersed in a 1% aqueous acetic acid solution. Thedispersion was heated at 100° C. for 2 hours to removeliposaccharide-linked oligosaccharides (Galanos C., Eur. J. Biochem.,24, 116-122, 1971). The thus treated cells were washed and coated with 4μg of PIP3. The resulting suspension was used as the immunogen (forsingle-use) (Umeda M., J. Immun., 137, 3264-3269, 1986). PIP3 waschemically synthesized by the known method (Sawada T., Bioorganic &Medicinal Chemistry Letters, 5, 2263-2266, 1995). The immunogen wasinjected into Balb/c mice via the tail veins a few times every otherweek. In the mice with an increased antibody titer, the spleen cellswere fused with myeloma cells P3-X63-Ag.653 to produce hybridomas.

[0058] Following the HAT selection, hybridoma supernatants were screenedfor the antibodies in terms of the binding activity to PIP3. The bindingactivity of the antibodies was screened by liposome lysis assay. At thesame time, the antibodies that produced positive clones were tested forthe cross-reactivity by indirect ELISA.

[0059] In the liposome lysis assay, PIP3 (1%), phosphatidylcholine(40%), cholesterol (40%) and dicetyl phosphate (9%) were dissolved inchloroform and distilled to dryness under reduced pressure to prepare alipid film. After a highly concentrated aqueous solution of calcein(fluorescent dye) was added to the film, the mixture was vigorouslystirred to prepare calcein marker-enclosed multilamellar liposomes. Theculture supernatant was added to the liposomes together with thecomplement so that the antigen-antibody binding occurred to activate thecomplement and break the membrane. When the antibody bound to PIP3 ispresent in the culture supernatant, the highly concentrated calcein inthe liposomes is released so that the concentration of calcein isreduced, causing fluorescence to be emitted. By measuring thefluorescent intensity, the activity of the antibody with respect to theantigen was determined.

[0060] Indirect ELISA was performed as follows. First, a solution of 100ng/mL of PIP3 in 5 μg/mL of phosphatidylcholine (carrier) was charged inmicrotiter plates. It was allowed to stand overnight at room temperaturefor coating, then dried. The wells were then incubated in a blockingbuffer (1% bovine serum albumin (BSA), 10 mM Hepes-buffered saline (HBS,pH 7.6)) for 30 minutes at room temperature. The blockedantigen-sensitized plates were washed, sealed and stored in arefrigerator until use.

[0061] After 100 μL each of PI-4,5-P2, IP3, IP4 and IP6 (seriallydiluted to 10⁰ to 10⁴ ng/mL with 0.5% BSA/HBS) and 100 μL of hybridomasupernatants were added to the wells of the antigen-sensitized plates,incubation was carried out at room temperature for 2 hours. The reactionsolution was removed. After washing with HBS, a second antibody(alkaline phosphatase-labeled anti-mouse IgG3 or anti-mouse IgM; dilutedto {fraction (1/2000)} with 0.5% BSA/HBS) was added to the systemfollowed by incubation at room temperature for 2 hours. After completionof the reaction, the unreacted antibodies were removed. The wells wereagain washed with HBS, and p-nitrophenyl phosphate (PNPP) was added tothe wells to measure the activity of the alkaline phosphatase remainingin the wells. When the antibody contained in the culture supernatantswas specific to PIP3, the immune reaction between the antibody and PIP3proceeded without competitive inhibition by the various co-existingcompounds. Thus, the higher alkaline phosphatase activity is retained inthe solid phase. In contrast, when no antibody was contained in theculture supernatants or when the antibodies were cross-reactive with thevarious co-existing antigens, the number of antibodies that reacted withPIP3 on the solid phase was reduced due to competitive inhibition. As aresult, the alkaline phosphatase activity retained on the solid phasebecame lower. The results of the culture supernatant of hybridoma AP3-11obtained by ELISA are shown in FIG. 1. Since AP3-11 produces theantibody that reacts only with PIP3, a concentration-dependent decreasein binding percent (taking the absorbance obtained with the culturesupernatant alone as 100%) is observed only for PIP3.

[0062] By this screening, two clones that produced an antibody reactiveonly with PIP3 and two clones that produce an antibody reactive withboth PIP3 and PI-4,5-P2 were established. Designations of these clonesare summarized in the table below. The antigen-bound activity of AP3-16was calcium dependent. Among these clones, the reactivity was examinedin more detail with the antibody produced by AP3-11 that was specific toPIP3 and non-cross-reactive with PI-4,5-P2 (hereinafter AP3-11 refers tothe monoclonal antibody, unless otherwise indicated). Clone ClassSpecificity Note AP3-11 IgG3 PIP3 AP3-16 IgM  PIP3 Ca²⁺ dependent AP23-4IgG3 PIP3/PI-4,5-P2 AP23-5 IgG3 PIP3/PI-4,5-P2

EXAMPLE 2 Specificity of Anti-PIP3 Monoclonal Antibody AP3-11 (IgG3)

[0063] Among the antibodies obtained in Example 1, AP3-11 reacted onlywith PIP3 and showed the highest specificity to PIP3. The reactivity ofthis antibody was further analyzed by liposome lysis assay. Prior to theassay, pristane-treated mice were intraperitoneally inoculated withhybridoma AP3-11 . Immunoglobulin was purified from the ascetic fluid byammonium sulfate fractionation and used as monoclonal antibody AP3-11.The following lipid antigens were used for the liposome lysis assay.

[0064] phosphatidylcholine (PC)

[0065] phosphatidylserine (PS)

[0066] phosphatidic acid (PA)

[0067] phosphatidylinositol (PI)

[0068] cardiolipin (CL)

[0069] phosphatidylethanolamine (PE)

[0070] Liposomes prepared for the assay consisted of 50% cholesterol and40% phosphatidylcholine as the main constituents and 10% of variousdifferent lipids as the remaining constituents. The liposomes differentin composition were prepared in the same manner. The binding activity ofAP3-11 was assayed by varying the antibody concentration in 100⁰ to 10⁶pg/ML based on the liposomes. Lysis of the liposomes was observed to beconcentration-dependent only in the liposomes containing PIP3(PI-3,4,5-P3). In contrast, no lysis was observed in the liposomescontaining other membrane-constructing phospholipids such as PC, PS, PA,PI, CL or PE, even when using the solution of the highest antibodyconcentration (10⁶ pg/mL) prepared for the assay. The results are shownin FIG. 2. In FIG. 2, liposome lysis (%) refers to a percentage when thefluorescent intensity is made 100% when all of the liposomes reactedwere lysed. The results reveal that AP3-11 reacted only with PIP3 in thephospholipids. To obtain more detailed information on an epitoperecognized by AP3-11, the cross-reactivity of phosphatidylinositol withvarious phosphorylated antigen derivatives was examined.

[0071] Liposomes prepared for the assay consisted of 50% cholesterol,40% phosphatidylcholine, and 9% dicetyl phosphate as the mainconstituents and 1% of the following phosphorylated phosphatidylinositolderivatives as the remaining constituents. In addition to this assay,direct ELISA was performed using microtiter plates to which PIP3 insteadof the following compounds was immobilized.

[0072] PI-3,4,5-P3

[0073] PI-3,4-P2

[0074] PI-4,5-P2

[0075] PI

[0076] The assay was performed by varying the antibody concentrationfrom 10⁰ to 10⁶ pg/mL. As in the previous experiment, lysis of theliposomes was observed to be concentration-dependent in the liposomescontaining PIP3 (PI-3,4,5-P3). However, no reaction was observed in theliposomes containing PI or PI-4,5-P2, even when the solution of thehighest antibody concentration (10⁶ pg/mL) prepared for the assay wasused. For PI-3,4-P2, a cross-reactivity of approximately {fraction(1/100)} observed with PIP3 was noted. The results are shown in FIG. 3.Similar results were confirmed by direct ELISA as shown in FIG. 4. Thatis, the cross-reactivity of approximately {fraction (1/100)} as comparedto PIP3 was noted with PI-3,4-P2. The foregoing results reveal that thephosphate group at the 3-position of the inositol group plays the mostimportant role for antigen recognition of AP3-11, and that the phosphategroup at the 5-position also participates in the epitope configuration.

EXAMPLE 3 Epitope of Anti-PIP3 Monoclonal Antibody AP3-11 (IgG3)

[0077] To identify the recognition site of the AP3-11 antibody, indirectELISA was performed using as competitors the following inositolpolyphosphates (10⁰ to 10⁴ nM) having a similar configuration to theinositol group of PIP3. The inositol polyphosphates used are givenbelow.

[0078] inositol-1,4,5-triphosphate (IP3)

[0079] inositol-1,3,4,5-tetraphosphate (IP4)

[0080] inositol-1,2,3,4,5,6-hexaphosphate (IP6)

[0081] When free PIP3 was added as the competitor, absorbance wasreduced in a concentration-dependent manner by competition with thefixed PIP3. Other inositol polyphosphates, such as IP3, IP4 and IP6,were not affected, as shown in FIG. 5.

[0082] These results suggest that the glycerol backbone is involved inthe recognition site of the antibody. In order to verify whether theglycerol backbone constitutes the antibody recognition site, experimentswere performed using PIP3 analogs fixed onto beads. The PIP3 analogsused are IP3-APB and PIP3-APB shown by the formulae below. As shown inthe structural formulae, IP3-APB contains only the inositol structure ofPIP3, and PIP3-APB contains both the inositol ring and a part of theglycerol backbone of PIP3.

[0083] Individual PIP3 analogs were fixed onto beads to react withAP3-11. After the beads were electrophoresed by SDS-PAGE, the beads werestained with CBB to detect the heavy chains of the antibody. AP3-11bound to neither the beads themselves nor the IP3-APB-bound beads butbound to the PIP3-APB beads. When synthetic PIP3 was added as acompetitor upon mixing the PIP3-APB beads and the antibody, the bindingto the beads was inhibited, as shown in FIG. 6. These results indicatethat AP3-11 recognizes as the epitope the portion shown in FIG. 7,containing the glycerol backbone of PIP3, and that the phosphate groupat the 3-position of the inositol ring is extremely important.

[0084] The experimental results obtained with monoclonal antibody AP23-4that reacted both with PIP3 and with PI-4,5-P2 are also shown in FIG. 6.AP23-4 was observed to bind to the IP3-APB beads. It is thus consideredthat AP23-4 recognizes the antigen only by its inositol ring.

[0085] Since the absorbance reduction depended on the concentration ofPIP3 added, the antibody of the present invention makes the immunoassaypossible, based on the competitive reaction of PIP3. Furthermore, theantibody of the present invention is not affected by various othercompounds having similar configurations. Thus, the present invention canprovide a simple assay system which is excellent in specificity to PIP3.

EXAMPLE 4 Identification of Hypervariable Regions (CDR) of Anti-PIP3Monoclonal Antibody

[0086] cDNA encoding the variable regions of the monoclonal antibodyAP23-11 of the present invention that specifically recognizes PIP3 wascloned. RNA extracted from the hybridomas was subjected to RT-PCR foramplification of cDNA encoding the variable regions, using signalpeptide and constant region sequences as primers. First, the hybridomaAP23-11 was incubated in DMEM/10% FCS to prepare poly A⁺ RNA.Single-stranded cDNA was synthesized from 5 μg of the poly A⁺ RNA. PCRwas performed for 30 cycles, one cycle consisting of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 2 minutes. Restrictionenzyme recognition sites corresponding to the cloning sites ofpBluescript, which is a cloning vector, are provided at the 5′ end ofthe primer used.

[0087] A band of about 400 bp was isolated from the amplificationproduct by agarose gel electrophoresis and inserted into the cloningvector pBluescript. After the cloning, the vector was recovered. Thenucleotide sequence of the insert was confirmed by the dideoxy methodusing the vector primer and [alpha-32] dATP (F. Sanger, Science, 214,1205-1210, 1981).

[0088] The amino acid sequence the thus obtained gene encodes wasdeduced, and hypervariable regions CDR were identified by the ChothiaNumbering Scheme(http://www.biochem.ucl.ac.uk/-martin/abs/GeneralInfo.html#kabatnum,Al-Lazikani et al., J. Molec. Biol., 273, 927-948, 1997). The amino acidsequences constructing the thus identified variable regions of the heavychains, light chains, and CDRs are shown in FIG. 8. The amino acidresidues marked with the arrow in FIG. 8 hardly appear at thesepositions in conventional antibodies, according to the antibodydatabase. The amino acid residues located in the CDRs thus seem toparticipate in the specificity. In order to obtain mutants of the aminoacid sequences, the sequence containing the amino acid residues shouldbe selected.

What is claimed is:
 1. An antibody specifically recognizingphosphatidylinositol-3,4,5-triphosphate.
 2. An antibody of claim 1,wherein the antibody is a monoclonal antibody.
 3. An antibody of claim2, which recognizes an antigenic determinant comprising an inositolgroup and a glycerol backbone inphosphatidylinositol-3,4,5-triphosphate.
 4. An antibody of claim 1,which is non-cross-reactive with phosphatidylinositol-4,5-diphosphate.5. A variable region of immunoglobulin heavy chain specifically bindingto phosphatidylinositol-3,4,5-triphosphate, comprising an amino acidsequence shown by SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 2in which one or more amino acid residues have been substituted, deletedor added.
 6. A variable region of immunoglobulin light chainspecifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO: 4 or an amino acidsequence of SEQ ID NO: 4 in which one or more amino acid residues havebeen substituted, deleted or added.
 7. CDR1 in immunoglobulin heavychains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO: 5 or an amino acidsequence of SEQ ID NO:5 in which one or more amino acid residues havebeen substituted, deleted or added.
 8. CDR2 in immunoglobulin heavychains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO:6 or an amino acidsequence of SEQ ID NO:6 in which one or more amino acid residues havebeen substituted, deleted or added.
 9. CDR3 in immunoglobulin heavychains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO:7 or an amino acidsequence of SEQ ID NO:7 in which one or more amino acid residues havebeen substituted, deleted or added.
 10. CDR1 in immunoglobulin lightchains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO:8 or an amino acidsequence of SEQ ID NO:8 in which one or more amino acid residues havebeen substituted, deleted or added.
 11. CDR2 in immunoglobulin lightchains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO:9 or an amino acidsequence of SEQ ID NO:9 in which one or more amino acid residues havebeen substituted, deleted or added.
 12. CDR3 in immunoglobulin lightchains specifically binding to phosphatidylinositol-3,4,5-triphosphate,comprising an amino acid sequence shown by SEQ ID NO:10 or an amino acidsequence of SEQ ID NO:10 in which one or more amino acid residues havebeen substituted, deleted or added.
 13. An immunogen composition for usein producing an antibody specifically recognizingphosphatidylinositol-3,4,5-triphosphate, comprising a mixture of a deadSalmonella cell as an adjuvant andphosphatidylinositol-3,4,5-triphosphate.
 14. A method for producing anantibody specifically recognizingphosphatidylinositol-3,4,5-triphosphate, which comprises immunizing animmunogen composition comprising a mixture of a dead Salmonella cell asan adjuvant and phosphatidylinositol-3,4,5-triphosphate.
 15. Animmunoassay method which comprises the steps of reacting the antibodyspecifically recognizing phosphatidylinositol-3,4,5-triphosphate or avariable region thereof with phosphatidylinositol-3,4,5-triphosphatepresent in a sample, and detecting the binding based on an immunologicalreaction between the antibody or a variable region thereof and thetriphosphate.
 16. An immunoassay method of claim 15, which comprisesobserving the degree to which the immunological reaction between theantibody or a variable region thereof and an antigenic determinantrecognized thereby is inhibited byphosphatidylinositol-3,4,5-triphosphate present in a sample.
 17. A kitfor immunoassay for phosphatidylinositol-3,4,5-triphosphate comprisingthe antibody specifically recognizingphosphatidylinositol-3,4,5-triphosphate or a variable region thereof.